Phosphatidylserine delivery to endoplasmic reticulum-derived vesicles of plant cells depends on two biosynthetic pathways

Vesicles formed from endoplasmic reticulum (ER) by a cell-free system of leek cells (Allium porrum) are enriched in phosphatidylserine (PS), especially species containing very long chain fatty acids (VLCFA, at least 20 carbon atoms). In plant cells, PS is formed either by PS synthase or the serine exchange enzyme, although it is not known which pathway(s) contribute(s) to PS delivery in the ER-derived vesicles (EV), nor to what extent this occurs. Taking advantage of a cell-free system, we have shown that PS enrichment originates mainly from the serine exchange enzyme which is the only pathway that synthesizes the VLCFA-PS species. On the other hand, both enzymes synthesize PS with long chain fatty acids (up to 18 carbon atoms), but these species are given to the EV by PS synthase.     

Monensin blocks the transfer of very long chain fatty acid containing lipids to the plasma membrane of leek seedlings. Evidence for lipid sorting based on fatty acyl chain length.

Delivery of newly synthesized fatty acids and lipids to the plasma membrane in leek seedlings via the endoplasmic reticulum (ER)-Golgi apparatus pathway is primarily by bulk transport (without sorting). However, pulse-chase experiments revealed kinetics of transport of lipids with VLCFA (very long chain fatty acids having more than 18 carbon atoms) in favor of a preferential transfer of these molecules to the plasma membrane. Use of monensin showed the accumulation of lipids in the Golgi apparatus and a related decrease of the amount of lipids transported to the plasma membrane. Lipid and fatty acid analyses revealed that transport of VLCFA-containing phospholipids was most strongly inhibited by the monensin block. These results taken together with an inability of the plasma membrane to synthesize VLCFA support a role for the Golgi apparatus in VLCFA delivery to the plasma membrane and leads to the hypothesis of a sorting function as well, based on fatty acyl chain length.     

Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

ATP-Dependent Formation of Phosphatidylserine-Rich Vesicles from the Endoplasmic Reticulum of Leek Cells

Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morré, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625–637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPγ-S prevents vesicle formation. These vesicles correspond to small structures (70–80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells.     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Inhibition of the sterol pathway in leek seedlings impairs phosphatidylserine and glucosylceramide synthesis but triggers an accumulation of triacylglycerols

Like most higher plants, leek seedlings (Allium porrum L.) contain a mixture of Delta(5)-sterols in which sitosterol largely predominates. As previously reported (Plant Physiol., 117 (1998) 931), these compounds, which are synthesized at the endoplasmic reticulum level, were shown to be actively transported to the plasma membrane via a membrane-mediated process, together with phosphatidylserine (PS). In the present work, leek seedlings were allowed to germinate for 7 days in the presence of fenpropimorph, a sterol biosynthesis inhibitor. Such a treatment was found to trigger an almost complete replacement of the usual sterols by 9beta,19-cyclopropylsterols (mainly cycloeucalenol and 29-norcycloartenol). Extensive lipid analyses and labeling experiments with sodium [14C]acetate were performed to examine potential changes in the content and the rate of synthesis of the other lipid molecular species. The results indicate that the inhibition of the sterol pathway was accompanied by a severe decrease in PS and glucosylceramide synthesis as well as by a redirection of fatty acids toward the storage triacylglycerol pathway. Triacyglycerols are shown to accumulate concomitantly with a significant increase in intracellular lipid droplets in both aerial parts and roots of leek seedlings. Taken together, the present data emphasize that a coordinated regulation of the biosynthetic pathways of sterols and some specific lipid molecular species could take place during plant membrane biogenesis.     

Synthesis of phosphatidylserine in carrot cells cultured under carbon-source starvation

When carrot suspension cells were cultured on medium containing no carbon source (starvation), the levels of phosphatidylserine (PS) increased transiently 3-4 d after the initiation of starvation while levels of most other phospholipid (PL) species decreased. We previously reported that fatty acids of these PLs served as an alternative carbon source during starvation. The present study showed that cells possess two different biosynthetic pathways involving phosphatidylcholine (PC)/phosphatidylethanolamine (PE) exchange enzymes and PS synthase to synthesize PS. These activities peaked similarly 4 d after the initiation of starvation and coincided with the peak of PS level. The synthesis of serine was also significantly activated during starvation. The activity of phosphoserine aminotransferase (PSAT) which is involved in serine synthesis increased with a time course similar to that of the increase in the PS level. These observations suggest that the increase in PS level plays an important role in membranes which are degraded during starvation.     

Vesicular and non-vesicular lipid export from the ER to the secretory pathway

The endoplasmic reticulum is the site of synthesis of most glycerophospholipids, neutral lipids and the initial steps of sphingolipid biosynthesis of the secretory pathway. After synthesis, these lipids are distributed within the cells to create and maintain the specific compositions of the other secretory organelles. This represents a formidable challenge, particularly while there is a simultaneous and quantitatively important flux of membrane components stemming from the vesicular traffic of proteins through the pathway, which can also vary depending on the cell type and status. To meet this challenge cells have developed an intricate system of interorganellar contacts and lipid transport proteins, functioning in non-vesicular lipid transport, which are able to ensure membrane lipid homeostasis even in the absence of membrane trafficking. Nevertheless, under normal conditions, lipids are transported in cells by both vesicular and non-vesicular mechanisms. In this review we will discuss the mechanism and roles of vesicular and non-vesicular transport of lipids from the ER to other organelles of the secretory pathway.     

Fatty acid synthesis in mitochondria from Saccharomyces cerevisiae

The ability of purified mitochondria isolated from S. cerevisiae to synthesize fatty acids and especially very long chain fatty acids (VLCFA) has been investigated. The VLCFA synthesis requires malonyl-CoA as the C2 unit donor and NADPH as the reducing agent. Moreover the yeast mitochondrial elongase is able to accept either exogenous long chain fatty acyl-CoAs as substrates or elongate endogenous substrates. In the latter case, ATP is required for full activity. Besides this important VLCFA formation, the mitochondria from S. cerevisiae were also able to synthesize C16 and C18.     

Routing of the G Protein During Maturation of Festuca Leaf Streak Rhabdovirus in its Plant Host

By immunogold labelling the location of Festuca leaf streak virus glycoprotein (FLSV-G) was investigated in developing phloem and mature leaf parenchyma of Festuca gigantea infected with Festuca leaf streak virus (FLSV: Rhabdotiridae). In developing phloem cells, FLSV-G was detected in endoplasmic reticulum (ER). at perinuclear membranes, and in assembled virions, but neither in Golgi stacks and Golgi vesicles nor at the plasma membrane of infected cells. These results indicate that FLSV-G stays in the ER after transmembrane synthesis, and is not routed through the secretory pathway in F. gigantea. The membranous inclusions, present in infected mature leaf parenchyma cells were found to contain FLSV-G. It is suggested that the, virus-induced membranous inclusions have developed from FLSV-G-containing ER. The residence of FLSV-G in ER (present study) is in contrast to results with vesicular stomatitis virus (VSV; vertebrate rhabdovirus). Here the G protein is known to be routed to the plasma membrane through the secretory pathway.     

LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum

Background  

The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER.     

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.

Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.     

In vivo incorporation of acetate into the acyl moieties of polar lipids from etiolated leek seedlings

Seven-day-old leek seedlings actively synthesize lipids in vivo from [1-¹⁴C]acetate, both in the light and in the dark. In the dark, phospholipid synthesis is more effective than galactolipid synthesis. Whatever the time of acetate incorporation by the etiolated seedlings, very long chain fatty acids having from 20 to 26 carbon atoms are found in all the polar lipids, including the acyl-CoAs. All of the labelled very long chain fatty acids incorporated into the polar lipids are saturated. On the other hand, the labelled C₁₈-fatty acids are unsaturated in phospholipids and galactolipids and almost no label is found in the saturated or unsaturated C₁₈-fatty acids of the acyl-CoAs.     

H-Ras does not need COP I- or COP II-dependent vesicular transport to reach the plasma membrane

Although vesicular transport of the H-Ras protein from the Golgi to the plasma membrane is well known, additional trafficking steps, both to and from the plasma membrane, have also been described. Notably, both vesicular and nonvesicular transport mechanisms have been proposed. The initial trafficking of H-Ras to the plasma membrane was therefore examined in more detail. In untreated cells, H-Ras appeared at the plasma membrane more rapidly than a protein carried by the conventional exocytic pathway, and no H-Ras was visible on Golgi membranes in >80% of the cells. H-Ras was still able to reach the plasma membrane when COP II-directed transport was disrupted by two different mutant forms of Sar1, when COP I-mediated vesicular traffic from the endoplasmic reticulum to the Golgi was inhibited with brefeldin A, or when microtubules were disrupted by nocodazole. Although some H-Ras was present in the secretory pathway, protein that reached the membranes of the endoplasmic reticulum-Golgi intermediate compartment was unable to move further in the presence of nocodozale. These results identify an alternative mechanism for H-Ras trafficking that circumvents conventional COPI-, COPII-, and microtubule-dependent vesicular transport. Thus, H-Ras has two simultaneous but distinct means of transport and need not depend on vesicular trafficking for its delivery to the plasma membrane.     

Translocation of pyrene-labeled phosphatidylserine from the plasma membrane to mitochondria diminishes systematically with molecular hydrophobicity: implications on the maintenance of high phosphatidylserine content in the inner leaflet of the plasma membrane

To study the translocation of phosphatidylserine (PS) from plasma membrane to mitochondria, dipyrene PS molecules (diPyr(n)PS; n=acyl chain length) were introduced to the plasma membrane of baby hamster kidney cells (BHK cells) using either cyclodextrin-mediated monomer transfer or fusion of cationic vesicles. Translocation of diPyr(n)PS to mitochondria was assessed based on decarboxylation by mitochondrial PS decarboxylase (PSD). It was found that the rate of translocation diminishes systematically with acyl chain length (molecular hydrophobicity) of diPyr(n)PS. Using an in vitro assay, it was shown that the spontaneous translocation rates of long-chain diPyr(n)PS species are similar to those of common natural PS species, thus supporting the biological relevance of the data. These results, and other data arguing against the involvement of vesicular traffic and lipid transfer proteins, imply that spontaneous monomeric diffusion via the cytoplasm is the main mechanism of PS movement from the plasma membrane to mitochondria. This finding could explain why a major fraction of PS synthesized by BHK cells consists of hydrophobic species: such species have little tendency to efflux from the plasma membrane to mitochondria where they would be decarboxylated. Thus, adequate molecular hydrophobicity seems to be crucial for the maintenance of high PS content in the inner leaflet of the plasma membrane.     

Ethanol specifically alters the synthesis, acylation and transbilayer movement of aminophospholipids in rat-liver microsomes

By experimenting with the aminoalcohols [3-3H]serine and [2-14C]ethanolamine we have been able to relate the effects of ethanol upon the biosynthesis of radioactive aminophospholipids (APL) in rat-liver microsomes and their distribution within the bilayer. The translocation of newly synthesized molecules of aminophospholipids labeled with different fatty acids was also investigated. The synthesis of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by base-exchange reaction (BES) was inhibited in membranes exposed to ethanol in direct response to its concentration. In addition, 100 mM ethanol specifically inhibited the transport of newly synthesized PS to the inner leaflet, resulting in similar levels of PS in both leaflets of the bilayer. The inhibition of PE synthesis by ethanol caused a decrease in its distribution in both inner and outer leaflets. An in vitro study of the incorporation of radioactive palmitate and oleate into the PS and PE of microsomes incubated with ethanol showed a decrease in the radioactivity levels of PE, suggesting that ethanol was specifically inhibiting the corresponding acyltransferase. It specifically altered the transbilayer movement of newly acylated phospholipids, modifying the distribution of palmitoyl- and oleoyl-acylated PS and PE in both leaflets. These results demonstrate for the first time that ethanol interferes with both the synthesis and intramembrane transport of aminophospholipids in endoplasmic reticulum (ER) membranes. Bearing in mind that if a membrane is to function properly its structure must be in optimum condition; it is evident that the observed processes may be responsible to some degree for the pathophysiological effects of alcohol upon cells.     

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer’s disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.     

Brefeldin A induces a microtubule-dependent fusion of galactosyltransferase-containing vesicles with the rough endoplasmic reticulum

The fungal drug brefeldin A (BFA) has recently been found to induce a redistribution of medial- and cis-Golgi components to the endoplasmic reticulum (ER), raising the possibility of the existence of a retrograde pathway from the Golgi complex to the ER. Here, we demonstrate a BFA-induced reversible rearrangement of the trans-Golgi membrane protein galactosyltransferase (Gal-T) to the ER in HeLa cells. With immunofluorescence microscopy we have shown that BFA first caused a rapid change of Gal-T immunolabelling from a normal Golgi complex pattern to long and slender structures emanating from the cell centre and co-localizing with tubulin. Then immunofluorescence became ER-like. This effect was not dependent on ongoing protein synthesis and was reversed to normal within 120 min after removal of the drug. Restoration of the Golgi complex after removal of brefeldin A was energy-dependent but not mediated by microtubules nor dependent on protein synthesis. BFA-induced backflow of Gal-T was inhibited by nocodazole, a microtubule-disrupting agent. Immunoelectron microscopy showed that BFA treatment resulted in the fusion of Gal-T-containing vesicles with the ER. Furthermore, sucrose gradient centrifugation showed a significant shift in density of mature Gal-T polypeptides upon BFA treatment: about 40% of the enzyme migrated from its original density (1.13 g/ml) to the density of rough ER (1.19 g/ml). Thus, BFA caused microtubule-dependent vesicular backflow from a trans-Golgi component to the ER followed by fusion of the Golgi-derived vesicles with the ER.     

Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.